Biomedical subjects

Waldemar Waldeck

Publications and source records attributed to Waldemar Waldeck.

10 recordsLinked to original sources

PubMed2007

Induced and repressed genes after irradiation sensitizing by pentoxyphylline.

Aim in cancer therapy is to increase the therapeutic ratio eliminating the disease while minimizing toxicity to normal tissues. Radiation therapy is a main component in targeting cancer. Radiosensitizing agents like pentoxyphylline (PTX) have been evaluated to improve radiotherapy. Commonly, cells respond to radiation by the activation of specific early and late response genes as well as by inhibition of genes, which are expressed under normal conditions. A display of the genetic distinctions at the level of transcription is given here to characterize the molecular events underlying the radiosensitizing mechanisms. The method of suppression subtractive hybridization allows the visualization of both induced and repressed genes in irradiated cells compared with cells sensitized immediately after irradiation. The genes were isolated by cDNA-cloning, differential analysis and sequence similarity search. Genes involved in protein synthesis, metabolism, proteolysis and transcriptional regulation were detected. It is important that genes like KIAA280, which were only known as unidentified EST sequences before without function, but inaccessible by array technology were recovered as functional genes. Database searches for PTX-induced genes detected a human mRNA completely unknown. In case of suppressed genes, we detected several mRNAs; one thereof shows homology to a hypothetical protein possibly involved in signal transduction. A further mRNA encodes the protein BM036 supposed to associate with the E2F transcription factor. A hypothetical protein H41 was detected, which may repress the Her-2/neu receptor influencing breast cancer, gliomas and prostate tumors. Radiation combined with PTX may lead to a better prognosis by down regulation of the Her-2/neu, which will be proven by clinical studies in the near future.

Cell Line, Tumor↗

PubMed2006

Delivery of substances and their target-specific topical activation.

Goal in pharmaceutical research is achievement of necessary drug concentrations in the target organ, effective treatment with safe delivery of genetic agents, while sparing normal tissue and minimizing side effects. A new "BioShuttle"-delivery system harbouring a cathepsin B cutting site, a nuclear address sequence and a functional peptide was developed and tumor cells were treated. Transport and subcellular activation were determined by confocal laser scanning microscopy permitting the conclusion: BioShuttle-conjugates prove as efficient tools for genetic interventions by selective and topical activation of therapeutic peptide precursors by enzymatic cleavage. As shown here for glioma cells and the cathepsin B cleavable site, living cells can be treated with high specificity and selectivity for diagnostic and therapeutic purposes.

Administration, Topical↗

PubMed2005

Two-hybrid fluorescence cross-correlation spectroscopy detects protein-protein interactions in vivo.

Fluorescence cross-correlation spectroscopy (FCCS) uses the correlated motion of two distinct fluorophores to detect their interaction. Whereas FCCS has been used with chemically or genetically labeled interaction partners in vitro, FCCS has never been demonstrated in vivo between two autofluorescent proteins. At least one reaction partner was always chemically labeled. Fos and Jun, two components of the AP-1 transcription factor, are known to exert their function as a dimer and can therefore serve as a reference for dimer formation. Expressing fusion proteins between Fos and the enhanced green fluorescent protein (EGFP), as well as Jun and the monomeric red fluorescent protein 1 (mRFP1) in HeLa cells, we show here, for the first time, in vivo FCCS detection of protein-protein interactions. The mobility of the dimerized species is slow, indicating that DNA-binding might stabilize dimerization. The technique has rich potential applications for the rapid screening of protein-protein interactions in vivo, which are able to clarify events during the whole life of cells.

Biophysics↗

PubMed2004

HPV18 E6 and E7 genes affect cell cycle, pRB and p53 of cervical tumor cells and represent prominent candidates for intervention by use peptide nucleic acids (PNAs).

Approximately 100% of cervical carcinomas are causally linked to infections with high-risk human papillomaviruses (HPVs), whose oncogenicity has been assigned to the continued expression of two early viral genes, E6 and E7. Reversal of the transformed phenotype by inhibiting E6/E7 gene expression therefore provides a suitable goal for tumor therapy. We established an application controlling the E6/E7 expression of the HPV type 18, by using viral gene directed peptide nucleic acids (PNAs). One consequence was the complete change in growth to a stagnated behavior of the HPV 18 positive HeLa-S cells. With flow cytometry, we investigated changes in the cell cycle and expression of the pRB (retinoblastoma) and p53 genes acting as antagonists to E6 and E7. We realized that application of PNAs via intracellular cleavable conjugated peptide carriers mediates specific inhibitory effects and we showed that the combined E6/E7-directed PNA-application mediated a clear morphological change from suspension to adherend state and the cells stopped growth. These data could demonstrate a promising approach for development of new 'anti-gene therapeutics' against papillomavirus-induced human cancers.

Algorithms↗

PubMed2003

Counting nucleosomes in living cells with a combination of fluorescence correlation spectroscopy and confocal imaging.

Although methods for light microscopy of chromatin are well established, there are no quantitative data for nucleosome concentrations in vivo. To establish such a method we used a HeLa clone expressing the core histone H2B fused to the enhanced yellow fluorescent protein (H2B-EYFP). Quantitative gel electrophoresis and fluorescence correlation spectroscopy (FCS) of isolated oligonucleosomes show that 5% of the total H2Bs carry the fluorescent tag and an increased nucleosome repeat length of 204 bp for the fluorescent cells. In vivo, the mobility and distribution of H2B-EYFP were studied with a combination of FCS and confocal imaging. With FCS, concentration and brightness of nascent molecules were measured in the cytoplasm, while in the nucleoplasm a background of mobile fluorescent histones was determined by continuous photobleaching. Combining these results allows converting confocal fluorescence images of nuclei into calibrated nucleosome density maps. Absolute nucleosome concentrations in interphase amount up to 250 microM locally, with mean values of 140(+/-28)microM, suggesting that a condensation-controlled regulation of site accessibility takes place at length scales well below 200 nm.

Flow Cytometry↗

PubMed2003

Intracellular visualization of prostate cancer using magnetic resonance imaging.

The term "molecular imaging" can be broadly defined as the in vivo characterization and measurement of biological processes at the cellular and molecular level. Is a gene expression magnetic resonance imaging (MRI) possible? Therefore, we have developed a novel intravital and intracellular MRI contrast agent composed of a gadolinium complex, an oligonucleotide sequence [peptide nucleic acid (PNA)], and a transmembrane carrier peptide that is composed of a peptide sequence similar to that of the homeodomain of the Antennapedia protein. The goal of our study was to determine whether this contrast agent could be accumulated in tumor cells in vitro (HeLa cells) and in vivo (Dunning R3327 AT1 rat prostate adenocarcinoma) and whether the specificity of the PNA for the up-regulated c-myc mRNA in the cell's cytoplasm would have an effect on contrast agent retention in the tumor cells. Using the c-myc-specific and a c-myc-nonspecific control PNA, an increase in signal intensity in the tumor cells was observed after 10 min in vitro and in vivo (maximum was reached in HeLa cells in vitro in 60 min, in Dunning R3327 AT1 rat prostate adenocarcinoma cells in vivo in 30 min). This increase of signal intensity could be maintained in vitro in HeLa cells for only 4 h and in Dunning R3327 AT1 rat prostate adenocarcinoma cells in vivo at least for 5 h by using the c-myc mRNA-specific PNA as a "retention" agent.

Adenocarcinoma↗

PubMed2003

Analyzing intracellular binding and diffusion with continuous fluorescence photobleaching.

Transport and binding of molecules to specific sites are necessary for the assembly and function of ordered supramolecular structures in cells. For analyzing these processes in vivo, we have developed a confocal fluorescence fluctuation microscope that allows both imaging of the spatial distribution of fluorescent molecules with confocal laser scanning microscopy and probing their mobility at specific positions in the cell with fluorescence correlation spectroscopy and continuous fluorescence photobleaching (CP). Because fluorescence correlation spectroscopy is restricted to rapidly diffusing particles and CP to slower processes, these two methods complement each other. For the analysis of binding-related contributions to mobility we have derived analytical expressions for the temporal behavior of CP curves from which the bound fraction and/or the dissociation rate or residence time at binding sites, respectively, can be obtained. In experiments, we investigated HeLa cells expressing different fluorescent proteins: Although enhanced green fluorescent protein (EGFP) shows high mobility, fusions of histone H2B with the yellow fluorescent protein are incorporated into chromatin, and these nuclei exhibit the presence of a stably bound and a freely diffusing species. Nonpermanent binding was found for mTTF-I, a transcription termination factor for RNA polymerase I, fused with EGFP. The cells show fluorescent nucleoli, and binding is transient. CP yields residence times for mTTF-I-EGFP of approximately 13 s.

Bacterial Proteins↗

PubMed2003

The enhancement of neutron irradiation of HeLa-S cervix carcinoma cells by cell-nucleus-addressed deca-p-boronophenylalanine.

Boron neutron capture therapy (BNCT) is an experimental treatment modality which depends on a sufficient cellular uptake of Boron ((10)B) followed by an exposure to a thermal neutron beam from a nuclear reactor. High energetic particles (4He and 7Li) are created during the neutron capture reaction and produce DNA damages, which lead to cell killing. Regarding BNCT, the short radiation range of He- and Li-particles is decisive for the distribution of (10)B. Until now, BNCT has been lacking for therapeutically effective concentrations of (10)B. Twenty-four hours after the combined use of our 'Bioshuttle'-p-borono-phenylalanine(10)-constructs ('Bioshuttle'-p-BPA(10)) and neutron-irradiation, an obvious reduction of the radiation-resistant HeLa-S cells could be observed. No cells were alive 72 h after the incubation with 'Bioshuttle'-p-BPA(10) followed by neutron irradiation. A post-mitotic cell death could be assumed based on flow cytometrical data.

Active Transport, Cell Nucleus↗

PubMed2002

CNN-Gd(3+) enables cell nucleus molecular imaging of prostate cancer cells: the last 600 nm.

Molecular imaging is defined as the characterization and measurement of biological processes at the cellular and molecular level. Molecular imaging, therefore, necessitates a sufficient amount of contrast agent within the cell. Consequently, we realized that the intracellular uptake and cell compartment specificity of the commonly used interstitial contrast agent gadolinium (Gd(3+)) with a cell-nucleus directed peptide module could be helpful. This modular molecule is characterized by a Gd(3+)-complex module that is bound to a transmembrane transport unit (TPU) of human origin and further to a nucleus-directed address module (nuclear localization sequence) resulting in a specific cell nucleus-directed nuclear localization sequence-conjugated Gd(3+)-complex (CNN-Gd(3+)-complex). By use of magnetic resonance imaging, Gd(3+) was detected within DU-145 prostate cancer cells after only 10 min. The nuclear localization was confirmed with confocal laser scanning microscopy. The resulting MRI signal enhancement only slightly decreased over the next 48 h compared with an absolute loss of signal enhancement after only 8 h when a random target sequence was used. Therefore, our method seems promising for in vivo application in molecular imaging.

Amino Acid Sequence↗

PubMed2002

A biological transporter for the delivery of peptide nucleic acids (PNAs) to the nuclear compartment of living cells.

To facilitate nuclear delivery of biomolecules we describe the synthesis of a modular transporter bearing a cellular membrane transport peptide (pAntp) and, as a cargo, a 16-mer peptide nucleic acid (PNA) covalently linked to a nuclear localisation signal (NLS[SV40-T]). Transport peptide and PNA are connected via N-terminal activated cysteine to form cleavable disulphide bonds. Internalization and subsequent delivery of PNA to the nucleus was verified in living and fixed cells by confocal laser scanning microscopy (CLSM) and fluorescence correlation spectroscopy (FCS). Double-labelling experiments indicate the cytoplasmic cleavage of the two modules and the effective nuclear import of the chromophore-tagged cargo. A non-degradable linker between transport module and cargo as well as a construct without NLS did not enable nuclear PNA import under the described experimental conditions. FCS-measurements revealed that most of the PNAs delivered into the cytoplasm by the modular transporter are anchored or encapsulated, indicating that intracellular transport of these compounds is not governed by molecular diffusion. Our results clearly demonstrate efficient compartment-directed transport using a synthetic, non-toxic modular transporter in living cells.

Active Transport, Cell Nucleus↗